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| Cloning and Prokaryotic Expression of F Gene of Peste des petits ruminants virus GS Strain and Preparation of Polyclonal Antibody |
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Abstract Peste des petits ruminants (PPR) is a disease of major economic importance and imposes a significant constraint upon sheep(Ovis aries) and goat (Capra hircus) production owing to its high mortality rate. It is an acute, highly contagious and frequently fatal disease of sheep and goats caused by Peste des petits ruminants virus (PPRV), a member of genus Morbillivirus of family paramyxoviridae. In order to study the F gene sequence of PPRV GS strain and the immunogenicity of its prokaryotic expression products, the primers were designed according to the F gene sequence of PPRV Nigeria 75/1 strain in GenBank(GenBank No. HQ197753), and the F gene of PPRV GS strain was amplified by RT-PCR. Based on sequencing and sequence analysis, the primers were designed and the Fa fragment of F gene without the signal peptide and transmembrane domain was amplified. Then the recombinant plasmid pET-PPRV-Fa was constructed through cloning Fa fragment into pET-32a(+) and the recombinant plasmid was transformed into Escherichia coli Transetta (DE3). After induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), the recombinant protein was expressed, and the New Zealand rabbit (Oryctolagus cuniculus) was immunized with purified protein, then the polyclonal antibody against PPRV fusion protein was prepared. Subsequently, the analysis of its immunogenicity was accomplished using indirect enzyme-linked immunosorbent assay (ELISA) and Western blot. The results showed that the complete CDS of F gene (GenBank No. KX822738) from PPRV GS strain was 1 641 bp encoding a 546 amino acids protein. The Fa fragment of F gene was successfully expressed in E. coli, whose molecular weights approximately was 59 kD and mainly existed in the form of inclusion body. The indirect ELISA and Western blot results showed that the prokaryotic expression products of Fa gene of PPRV GS strain had good immunogenicity. In conclusion, the research results provide theoretical basis for the study on the function of fusion protein from PPRV GS strain, and for developing a rapid diagnosis method.
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Received: 18 October 2016
Published: 02 March 2017
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